M. Heather West Greenlee

Differential localization of SNARE complex proteins SNAP‐25, syntaxin, and VAMP during development of the mammalian retina

SNARE complex proteins have critical functions during regulated vesicular release of neurotransmitter. In addition, they play critical roles during neurite outgrowth and synaptogenesis. Although it is clear that the function of any one SNARE complex protein during release of neurotransmitter is dependent on its association with other members of the complex, it is less certain whether their function during development and differentiation is dependent on interaction with one another. Previously, we have observed transient high levels of SNARE complex protein SNAP‐25 in developing cholinergic amacrine cells (West Greenlee et al. [1998] J Comp Neurol 394:374–385). In addition, we detected, high levels of SNAP‐25 in developing and mature photoreceptors. To better understand the functional significance of these high levels of SNAP‐25 expression, we used immunocytochemistry to examine the developmental expression of the three members of the SNARE complex, SNAP‐25, Syntaxin, and vesicle associated membrane protein (VAMP/also Synaptobrevin). Our results demonstrate that the high levels of SNAP‐25 in cholinergic amacrine cells and photoreceptors are not accompanied by the same relatively high levels of other SNARE complex proteins. These results suggest that high levels of SNAP‐25 in specific cell types may function independently of association with Syntaxin and VAMP. In this analysis, we characterized the changing patterns of immunoreactivity for the three SNARE complex proteins during the development and differentiation of the mammalian retina. We have compared the pattern of expression of the core SNARE complex proteins in the Brazilian opossum, Monodelphis domestica, and in the rat and found common patterns of expression between these diverse mammalian species. We observed temporal differences in the onset of immunoreactivity between these three proteins, and differences in their localization within synaptic layers in the developing and mature mammalian retina. This study is the first to characterize the changing expression patterns of the three SNARE complex proteins in the developing central nervous system. The differential distribution of SNAP‐25, Syntaxin, and VAMP may indicate additional roles for these proteins during vesicle trafficking events, which are independent of their association with one another. J. Comp. Neurol. 430:306–320, 2001.

Clinical and Pathologic Features of H-Type Bovine Spongiform Encephalopathy Associated with E211K Prion Protein Polymorphism

The majority of bovine spongiform encephalopathy (BSE) cases have been ascribed to the classical form of the disease. H-type and L-type BSE cases have atypical molecular profiles compared to classical BSE and are thought to arise spontaneously. However, one case of H-type BSE was associated with a heritable E211K mutation in the prion protein gene. The purpose of this study was to describe transmission of this unique isolate of H-type BSE when inoculated into a calf of the same genotype by the intracranial route. Electroretinograms were used to demonstrate preclinical deficits in retinal function, and optical coherence tomography was used to demonstrate an antemortem decrease in retinal thickness. The calf rapidly progressed to clinical disease (9.4 months) and was necropsied. Widespread distribution of abnormal prion protein was demonstrated within neural tissues by western blot and immunohistochemistry. While this isolate is categorized as BSE-H due to a higher molecular mass of the unglycosylated PrPSc isoform, a strong labeling of all 3 PrPSc bands with monoclonal antibodies 6H4 and P4, and a second unglycosylated band at approximately 14 kDa when developed with antibodies that bind in the C-terminal region, it is unique from other described cases of BSE-H because of an additional band 23 kDa demonstrated on western blots of the cerebellum. This work demonstrates that this isolate is transmissible, has a BSE-H phenotype when transmitted to cattle with the K211 polymorphism, and has molecular features that distinguish it from other cases of BSE-H described in the literature.

Aligning biomolecular networks using modular graph kernels

Comparative analysis of biomolecular networks constructed using measurements from different conditions, tissues, and organisms offer a powerful approach to understanding the structure, function, dynamics, and evolution of complex biological systems. We explore a class of algorithms for aligning large biomolecular networks by breaking down such networks into subgraphs and computing the alignment of the networks based on the alignment of their subgraphs. The resulting subnetworks are compared using graph kernels as scoring functions. We provide implementations of the resulting algorithms as part of BiNA, an open source biomolecular network alignment toolkit. Our experiments using Drosophila melanogaster, Saccharomyces cerevisiae, Mus musculus and Homo sapiens protein-protein interaction networks extracted from the DIP repository of protein-protein interaction data demonstrate that the performance of the proposed algorithms (as measured by % GO term enrichment of subnetworks identified by the alignment) is competitive with some of the state-of-the-art algorithms for pair-wise alignment of large protein-protein interaction networks. Our results also show that the inter-species similarity scores computed based on graph kernels can be used to cluster the species into a species tree that is consistent with the known phylogenetic relationships among the species.

Amyloid-beta peptide affects viability but not differentiation of embryonic and adult rat hippocampal progenitor cells

The neurological deficits that are characteristic of Alzheimers Disease (AD) are ultimately a result of neuronal loss in distinct anatomical regions of the brain. This neuronal loss is thought to be due, in large part to the presence of the neurotoxic beta-amyloid (Abeta) deposits, that are characteristic of the AD brain. Transplantation therapy, in which neural stem cells (NSCs) or neural progenitor cells (NPCs) are introduced into damaged regions of the brain and induced to differentiate into replacement neurons, has been proposed as a possible therapeutic approach to treat AD. However, in the AD brain Abeta plaques, which remain in the area of neuronal degeneration, may affect the viability or differentiation potential of transplanted NSCs. Currently there is contradictory evidence concerning the effect of Abeta on NSCs. To further investigate the effect of Abeta on NSCs, we compared the mitochondrial function, proliferation and cellular differentiation of two populations of hippocampal NSCs (embryonic and adult derived) after Abeta exposure. Our results highlight the heterogeneity between different populations of NSCs even when derived from the same brain region. Our data also demonstrate that while mitochondrial function of NSCs is affected by Abeta, their proliferation and differentiation are not significantly influenced. Considered with previous studies, our results suggest that while NSCs do respond to the presence of Abeta, proliferation and differentiation of certain populations are not affected. Further study of the differences between susceptible vs. resistant populations of NSCs may provide crucial clues for the development of effective therapies to combat AD.

Neurologic Manifestations of Leishmania spp. Infection

When listing common clinical signs of the spectra of Leishmania-derived diseases, neurologic malfunctions are not commonly included. Despite this, there are multiple reported instances both in human and veterinary medicine where neurologic manifestations, whether central or peripheral, are described. In this review, we describe neurologic manifestations seen during infection with Leishmania spp. with some discussion of the implicit effect of inflammation on the blood brain barrier in both medical and veterinary cases. Taken together, the material discussed here suggests that in patients from Leishmania-endemic areas, when observing neurologic symptoms, causation secondary to infection with Leishmania spp. should be highly considered.

The Transmissible Spongiform Encephalopathies of Livestock

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal protein-misfolding neurodegenerative diseases. TSEs have been described in several species, including bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, chronic wasting disease (CWD) in cervids, transmissible mink encephalopathy (TME) in mink, and Kuru and Creutzfeldt-Jakob disease (CJD) in humans. These diseases are associated with the accumulation of a protease-resistant, disease-associated isoform of the prion protein (called PrP(Sc)) in the central nervous system and other tissues, depending on the host species. Typically, TSEs are acquired through exposure to infectious material, but inherited and spontaneous TSEs also occur. All TSEs share pathologic features and infectious mechanisms but have distinct differences in transmission and epidemiology due to host factors and strain differences encoded within the structure of the misfolded prion protein. The possibility that BSE can be transmitted to humans as the cause of variant Creutzfeldt-Jakob disease has brought attention to this family of diseases. This review is focused on the TSEs of livestock: bovine spongiform encephalopathy in cattle and scrapie in sheep and goats.

Using Evolutionary Conserved Modules in Gene Networks as a Strategy to Leverage High Throughput Gene Expression Queries

Background Large-scale gene expression studies have not yielded the expected insight into genetic networks that control complex processes. These anticipated discoveries have been limited not by technology, but by a lack of effective strategies to investigate the data in a manageable and meaningful way. Previous work suggests that using a pre-determined seed-network of gene relationships to query large-scale expression datasets is an effective way to generate candidate genes for further study and network expansion or enrichment. Based on the evolutionary conservation of gene relationships, we test the hypothesis that a seed network derived from studies of retinal cell determination in the fly, Drosophila melanogaster, will be an effective way to identify novel candidate genes for their role in mouse retinal development. Methodology/Principal Findings Our results demonstrate that a number of gene relationships regulating retinal cell differentiation in the fly are identifiable as pairwise correlations between genes from developing mouse retina. In addition, we demonstrate that our extracted seed-network of correlated mouse genes is an effective tool for querying datasets and provides a context to generate hypotheses. Our query identified 46 genes correlated with our extracted seed-network members. Approximately 54% of these candidates had been previously linked to the developing brain and 33% had been previously linked to the developing retina. Five of six candidate genes investigated further were validated by experiments examining spatial and temporal protein expression in the developing retina. Conclusions/Significance We present an effective strategy for pursuing a systems biology approach that utilizes an evolutionary comparative framework between two model organisms, fly and mouse. Future implementation of this strategy will be useful to determine the extent of network conservation, not just gene conservation, between species and will facilitate the use of prior biological knowledge to develop rational systems-based hypotheses.

Changes in Retinal Function and Morphology Are Early Clinical Signs of Disease in Cattle with Bovine Spongiform Encephalopathy

Bovine spongiform encephalopathy (BSE) belongs to a group of fatal, transmissible protein misfolding diseases known as transmissible spongiform encephalopathies (TSEs). All TSEs are caused by accumulation of misfolded prion protein (PrPSc) throughout the central nervous system (CNS), which results in neuronal loss and ultimately death. Like other protein misfolding diseases including Parkinson’s disease and Alzheimer’s disease, TSEs are generally not diagnosed until the onset of disease after the appearance of unequivocal clinical signs. As such, identification of the earliest clinical signs of disease may facilitate diagnosis. The retina is the most accessible part of the central nervous system, and retinal pathology in TSE affected animals has been previously reported. Here we describe antemortem changes in retinal function and morphology that are detectable in BSE inoculated animals several months (up to 11 months) prior to the appearance of any other signs of clinical disease. We also demonstrate that differences in the severity of these clinical signs reflect the amount of PrPSc accumulation in the retina and the resulting inflammatory response of the tissue. These results are the earliest reported clinical signs associated with TSE infection and provide a basis for understanding the pathology and evaluating therapeutic interventions.

Glucose uptake in PC12 cells: GLUT3 vesicle trafficking and fusion as revealed with a novel GLUT3-GFP fusion protein

The distribution of glucose transporters at the cell surface has a major impact on cellular glucose uptake. In muscle cells and adipocytes, this distribution is under the control of insulin; however, neuronal glucose uptake is not acutely regulated by insulin. Factors that affect the translocation of the neuronal glucose transporter isoform GLUT3 vesicles to and their fusion with the plasma membrane are not well understood. We report that GLUT3 in PC12 cells colocalizes with SNARE complex proteins SNAP‐25 and syntaxin 1, suggesting that fusion of GLUT3‐containing vesicles with the plasma membrane is mediated by these proteins. In addition, it seems that GLUT3 vesicle fusion is regulated, as depolarization increases GLUT3 insertion into the plasma membrane. To study the dynamics of GLUT3 vesicle trafficking, we have created a GLUT3‐GFP fusion protein that is easily expressed in PC12 cells. Trafficking of GLUT3‐GFP seems normal, as 1) its distribution is similar to endogenous GLUT3, 2) GLUT3‐GFP containing vesicles fuse with the plasma membrane evidenced by labeling of the fusion protein with an antibody directed against the exofacial epitope of GLUT3, and 3) glucose uptake is similar to PC12 cells not transfected with GLUT3 fusion protein. These studies are the first to examine GLUT3 trafficking and fusion in PC12 cells, and establish a model system to study regulation of the neuronal glucose transporter.

Expression of SNAP-25 during mammalian retinal development: thinking outside the synapse.

The SNARE complex is the core machinery required for vesicle fusion events. Numerous structural, functional, and genetic studies have led to a better understanding of mechanisms that regulate vesicle fusion events during neural development. Studies using the mammalian retina as a model system have increased our understanding of the dynamic patterns of expression of SNARE proteins. In particular, the SNARE complex protein SNAP-25 is expressed in a dynamic fashion during the development of cholinergic amacrine cells in a number of mammalian species. SNAP-25 is also likely to play a crucial role during the development of vertebrate photoreceptors. The integration of comparative studies examining SNARE proteins, such as SNAP-25, provides a powerful approach for the study of CNS development.